Fluorescent dyes are widely used for labeling, detecting, and/or quantifying components in a sample. The various approaches used for such detection and/or quantification include fluorescence microscopy, fluorescence immunoassay, flow cytometric analysis of cells, and various other applications. In general, for many applications that utilize fluorescent dyes as detection tools, it is necessary to conjugate the fluorescent dye with a ligand such as a protein, antibody, enzyme, nucleotide, nucleic acid, and other biological and non-biological molecules to make a dye-labeled ligand. The dye-labeled ligand is an important reagent that confers specificity for a subsequent biochemical interaction in which the fluorescent dye provides a method for detection and/or quantification of the interaction.
The choice in fluorescent dyes is particularly important in applications that utilize multiplex, multicolor analysis, such as fluorescence microscopy, fluorescence immunoassay, flow cytometry, and various other applications. Notably, certain detection applications require an ultraviolet excitable fluorophore having a specific excitation range.
Specifically, there is a need for a fluorescent dye that can be efficiently excited by the 405 nm violet laser in multi-color flow cytometry instruments. The target dye for such applications should essentially have the following features: 1) a maximum in its excitation spectrum near 405 nm, 2) a strong spectrally resolvable emission maximum, 3) a large Stokes' shift, preferably, at least 50 nm, and 4) the ability of the fluorescent dye to couple to a biomolecule through a reactive group. Heretofore, there is a shortage of ultraviolet excitable fluorophores that can be conjugated to different ligands to provide dye-labeled reagents having optically and electronically separable fluorescence spectral properties.
The fluorescent dyes of the invention are structurally similar to firefly luciferin compounds. Such compounds have been previously used as chemiluminescent reagents whereby light is generated by the oxidative catalysis of luciferin. Luciferin, a substrate for the enzyme luciferase, is oxidized in the presence of luciferase to produce oxyluciferin and energy which is released in the form of light. For these types of assays, the luciferase-luciferin reaction provides the basis for simple, rapid, and sensitive assays for a wide range of substances (Karicka, L J., Analytical Biochemistry, 175, 14-21 (1988)).
While such chemiluminescent dyes based on the structure of firefly luciferin have gained widespread use, such use does not evidence the capability of compounds having a structure similar to that of firefly luciferin to function as fluorescent dyes excitable within a specific part of the ultraviolet spectrum. Such ultraviolet excitable fluorescent dyes are particularly advantageous for a wide range of applications, including without limitation, applications that utilize multiplex, multicolor fluorescence analysis.